A Quantitative Approach to Estimate both Essential and Non-essential Elements in Some Commercial Samples of Triphala churna by using WD-XRF Spectrometry

Introduction: The need for quality control of herbal drugs is in demand in order to ensure the purity, safety and efficacy of herbal products. A total of 19 elements including essential and non-essential elements were characterized in five commercial samples of Triphala churna using WD-XRF spectrometry. Method: The WD-XRF method was validated for each element by a pre-calibrated program using five Chinese certified reference materials of vegetable standards (NCS ZC73012, NCS ZC73013, NCS ZC73017, NCS ZC85006 and NCS DC73348). Results: The following elements were detected in all the samples out of 19 elements tested with increasing order of concentrations (mg/kg): Cr (3) < Cu (7) < Ba (24) < Zn (31) < Pb (46) < Mn (57) < S (700) < Na (1064) < Mg (1250) < Fe (1329) < P (1400) < Cl (2960) < Ca (3110) < Si (4350) < K (15130). Lead (41-46 mg/kg), a nonessential element was found above its PDE limit (≤ 10 mg/kg). Conclusion: WD-XRF method was found simple, rapid, reliable and non-destructive technique to investigate the elemental concentrations in herbal drugs.


INTRODUCTION
Ayurvedic medicines are one of the important ancient medical systems of India.About 80% of the world population still primarily rely on herbal drugs. 1 In India, Ayurvedic medicines were widely considered due to affordability and availability in local markets.Also, the demand for Ayurveda drugs in Western market has increased tremendously which draw attention to the need of quality of Ayurvedic products.Especially, the presence of heavy metals at high concentrations in these drugs are potentially harmful to consumers and causing severe adverse health effects.Hence, the screening of these herbal drugs is given preference in scientific studies and the WHO in number of resolutions has emphasized the need of quality control of plant products by using modern techniques and suitable analytical methods. 2 Therefore, the present study was focused to carry out to analyse essential and non-essential elements using WD-XRF spectrometry in five commercially important Triphala churna drugs.Triphala churna is a mixture of three dried fruits (Haritaki, Bibhitaki and Amalaki) in equal proportions (1:1:1) and a variety of therapeutic applications have been described in ISM. 3 for Chemical Analysis, Central Research Laboratory, GITAM University, Visakhapatnam, India.The brand names were blinded and coded in sequential order i.e.TPC1, TPC2, TPC3, TPC4 and TPC5.

Experimental and Instrumental Details
Pellets of each drug sample and standard were made by taking three grams of powder sample in an aluminum cup and pressing using a hydraulic press (HERZOG, TP40/2D) at 15 tons to obtain a disc shape with 40 mm diameter.These pellets were subjected to WD-XRF spectrometer and characterized the elemental composition as described by Swamy and Sivanarayanan (2014). 4

RESULTS AND DISCUSSION
In the present study, the levels of 19 essential and non-essential elements such as Na, Mg, Al, Si, P, S, Cl, K, Ca, Cr, Mn, Fe, Cu, Zn, As, Ba, Cd, Hg and Pb were determined using WD-XRF spectrometry in five samples of Triphala churna are summarized in Table 1 and they are the mean of three replicates.The WD-XRF method was validated for each element and good agreements were achieved between present values and published data. 4The available specific standards for few trace elements (Pb ≤ 10 ppm, Cd ≤ 0.3 ppm, As ≤ 10 ppm and Hg ≤ 1 ppm) by WHO 5 and tolerance limits of elements in food or 4350 mg/kg, an essential mineral required for human body to grow and maintain strong bones, hair, fingernails, connective tissues, skin, blood vessels and healthy artery walls. 10,11It helps to counter the effects of high accumulation of Al in the body, but, an excess of Si may cause irreversible kidney damage.The other elements that are analyzed, As, Cd, Hg and Pb are not useful role in human physiology and therefore considered to be non-essential elements.Of these, presence of Pb was detected in all market samples tested in the present study with a concentration range of 41 -46 mg/kg (Table 1), which is almost 4 folds higher than its PDE (≤ 10 mg/kg).In fact, Pb existence in herbal drugs above its permissible limit is predictable and reported in many earlier studies.However, high dose of 'Pb' detected in current study in all samples may cause harmful health effects 12 and alarming the safety concern of these herbal products.As, Cd and Hg were not detected due to they maybe present below its detection limit of XRF and in such cases, XRF should be used in conjunction with a more sensitive technique, AAS / ICP.

Comparison of XRF data with ICP Data
The WD-XRF technique has adequate sensitivity to determine essential as well as trace elements and was well proved in earlier studies. 4However, the results obtained in the present study by WD-XRF spectrometry were cross checked with available ICP-MS data (Table 2). 3 The Cu mean values determined by the WD-XRF method was slightly higher than that determined by the ICP-MS method with good RSD (3-28%) and only TPC1 sample was noticed with a higher ICP value with 28% RSD.A significant difference was observed for Zn i.e. almost two fold higher XRF values were noticed when compared with ICP data with 32-40% RSD.Agreement, typically 22-50% (RSD) was noticed between methods WD-XRF and ICP-MS for Cr.The mean values determined by the WD-XRF method was noticed to be slightly higher for elements Ba, Ca, Fe, K, Mg, Mn, Na when compared with reported ICP-MS data and they are close to each other with good agreement i.e.RSD (%), 19-25, 8-10, 12-19, 4-12, 14-17, 16-20 and 22-25, respectively.These variations in elemental con-dietary intake published by CEC 6 are considered for quality regulation of Ayurvedic drugs tested in this study (Table 1).

Interpretation of WD-XRF data
Out of 19 elements analysed, Na, K, Mg, Ca, P, S and Cl are essential nutrients and they were present in substantial quantities in all remedies and most of them were within tolerable upper intake level (Table 1).Elements such as Ba, Cr, Cu, Al, Mn, Fe, Zn and Si are considered as micronutrients and exhibit relatively low toxicity with high PDE. 7The concentration of 'Ba' in the present study was ranged from 20 to 24 mg/kg and it was within the permissible limits according to WHO guidelines (2004) i.e. the suggested mean daily intake of Ba from food, water and air is about 100 mg/kg. 8Trivalent Cr has a low level of toxicity at high concentrations, but, hexavalent Cr is more toxic and causes renal and hepatic necrosis and growth retardation. 6In the present study, Cr was found to be present in all samples ranging from 1 to 3 mg/kg, which is below the PDE limit i.e. 15 mg/kg as per USP guidelines (2009). 9In all the samples, Cu was detected within PDE limit with concentration between 5 to 7 mg/kg and Zn was ranged 27 to 31 mg/kg which is about 30 times lesser than the recommended PDE limit by CEC (Table 1).The concentration of 'Fe' in the present study was found within PDE limit in all the samples except TPC1 (1329 mg/kg, Table 1).The excess iron levels in the body can damages cells in the heart and liver which can cause cancer, diabetes, coma, metabolic acidosis, liver failure, cardiac failure, circulatory shock and long-term organ damages. 10An excess of Mn can lead to poor iron absorption and its deficiency can cause poor bone formation, affect fertility and ability of blood to clot. 10 In the present study, Mn was found below the PDE limit in all the samples within the range of 30 to 57 mg/kg (Table 1).Aluminum intake even in small amounts may lead to bio-accumulation in consumer's body, which is so difficult to get-rid-off and further, cumulative Al in the body can reduce the absorption of a number of elements including essential elements Fe, P, Ca, etc. 10,11 In the present study, Al was detected in all the samples within the range of 362 to 1140 mg/kg.Silicon was ranged from 1590 to

CONCLUSION
XRF is a non-destructive sample technique and the results obtained by WD-XRF spectrometry in present study were found to be compatible with reported ICP-MS data which suggests that the XRF technique can be a good choice for estimation of elemental concentrations in plant / herbal / Ayurvedic samples.

ACKNOWLEDGEMENT
The authors express their gratitude to the Vice Chancellor of GITAM University, Visakhapatnam, for encouragement and facilitating necessary *published data 3 ; SD: standard deviation; RSD: relative standard deviation congenial environment to carry out research work.Also, the authors thank the Head, Laboratory X-ray Crystallography, Indian Institute of Chemical Technology (CSIR), Hyderabad, India, for extending their constant support.

Table 1 : Levels of elements in various Triphala Churna drugs by WD-XRF Pharmacognosy
MS method3 with respect to XRF data, may be due to multi-elemental standards, different sample extraction procedures and various dilution concentrations are part of ICP analysis whereas XRF is a non-destructive technique.In hypothesis, a specific digestion method only recovers certain elements absolutely that are easily soluble and not structurally complicated, especially present in plant materials.In the opposite case, technique XRF also suffers from significant matrix effects present in plant materials.However, the XRF technique penetrates deeper into the sample bonds than the chemical extraction procedure for ICP-MS, may be showing higher concentrations for the same sample.